This project aims to determine the distribution of diffusible and structural components at chemical synapses. This work is significant because of the relationship between the localization and movement of such constituents and their role in synaptic transmission. To attain the resolution and sensitivty necessary to study synapses, this project combines several significant technological advances, including rapid freezing and cryosectioning of unfixed tissues, cryosectioning and immunocytochemical staining of sucrose-protected tissues, and quantitative, element-specific x-ray imaging and analysis in a specialized analytical electron microscope. Studies of the intracellular calcium distribution in the molecular layer of mouse cerebellum indicate that presynaptic calcium stores are not present and are not required for the activity of parallel fiber/Purkinje cell synapses. Membrane depolarization, however, is accompanied by the loading of extracellular calcium into organelles in the dendrites of Purkinje cells, demonstrating a requirement for calcium-handling organelles in postsynaptic elements. Analysis of calcium distribution in the synaptosomes from squid brain confirm the absence of presynaptic calcium stores in resting cholinergic terminals. This preparation is also being used, in conjunction with an antimony-labeled acetylcholine analog, to determine where ACh is taken up and stored in cholinergic synaptosomes. Correlative studies on structural aspects of synaptic function have shown that rapid freezing is essential for preserving the native organization of labile membrane structures such as vesicles. Immunocytochemical studied have supported the involvement of actin and brain spectrin in myelination, and provided an approach to determining the role of cytoskeletal proteins in the organization of brain synapses. Thus, this project has now begun to provide important information on the detailed relationship between the diffusible and structural components of synapses, and how these regulate synaptic activity.